RESUMEN
Cancer cells reprogram energy metabolism through metabolic plasticity, adapting ATP-generating pathways in response to treatment or microenvironmental changes. Such adaptations enable cancer cells to resist standard therapy. We employed a coculture model of estrogen receptor-positive (ER+) breast cancer and mesenchymal stem cells (MSC) to model interactions of cancer cells with stromal microenvironments. Using single-cell endogenous and engineered biosensors for cellular metabolism, coculture with MSCs increased oxidative phosphorylation, intracellular ATP, and resistance of cancer cells to standard therapies. Cocultured cancer cells had increased MCT4, a lactate transporter, and were sensitive to the MCT1/4 inhibitor syrosingopine. Combining syrosingopine with fulvestrant, a selective estrogen receptor degrading drug, overcame resistance of ER+ breast cancer cells in coculture with MSCs. Treatment with antiestrogenic therapy increased metabolic plasticity and maintained intracellular ATP levels, while MCT1/4 inhibition successfully limited metabolic transitions and decreased ATP levels. Furthermore, MCT1/4 inhibition decreased heterogenous metabolic treatment responses versus antiestrogenic therapy. These data establish MSCs as a mediator of cancer cell metabolic plasticity and suggest metabolic interventions as a promising strategy to treat ER+ breast cancer and overcome resistance to standard clinical therapies. IMPLICATIONS: This study reveals how MSCs reprogram metabolism of ER+ breast cancer cells and point to MCT4 as potential therapeutic target to overcome resistance to antiestrogen drugs.
Asunto(s)
Neoplasias de la Mama , Células Madre Mesenquimatosas , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Receptores de Estrógenos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Metabolismo Energético , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Microambiente TumoralRESUMEN
The peak prevalence of ESRD from glomerulosclerosis occurs at 70 to 79 years. To understand why old glomeruli are prone to failure, we analyzed the Fischer 344 rat model of aging under ad libitum-fed (rapid aging) and calorie-restricted (slowed aging) conditions. All glomerular cells contained genes whose expression changed "linearly" during adult life from 2 to 24 months: mesangial cells (e.g., MMP9), endothelial cells (e.g., ICAM and VCAM), parietal epithelial cells (e.g., ceruloplasmin), and podocytes (e.g., nephrin and prepronociceptin). Patterns of aging glomerular gene expression closely resembled atherosclerosis, including activation of endothelial cells, epithelial cells, and macrophages, as well as proinflammatory pathways related to cell adhesion, chemotaxis, blood coagulation, oxidoreductases, matrix metalloproteinases, and TGF-beta activation. We used a nonbiased data-mining approach to identify NFkappaB as the likely transcriptional regulator of these events. We confirmed NFkappaB activation by two independent methods: translocation of NFkappaB p50 to glomerular nuclei and ChIP assays demonstrating NFkappaB p50 binding to the kappaB motif of target genes in old versus young glomeruli. These data suggest that old glomeruli exhibit NFkappaB-associated up-regulation of a proinflammatory, procoagulable, and profibrotic phenotype compared with young glomeruli; these distinctions could explain their enhanced susceptibility to failure. Furthermore, these results provide a potential mechanistic explanation for the close relationship between ESRD and atherosclerotic organ failure as two parallel arms of age-associated NFkappaB-driven processes.
Asunto(s)
Coagulación Sanguínea , Inflamación/etiología , Glomérulos Renales/patología , FN-kappa B/fisiología , Factores de Edad , Animales , Fibrosis/etiología , Regulación de la Expresión Génica , Masculino , FN-kappa B/genética , Ratas , Ratas Endogámicas F344RESUMEN
Glomerular capillary filtration barrier characteristics are determined in part by the slit-pore junctions of glomerular podocytes. Protein tyrosine phosphatase receptor-O (PTPro) is a transmembrane protein expressed on the apical surface of podocyte foot processes. Tyrosine phosphorylation of podocyte proteins including nephrin may control the filtration barrier. To determine whether PTPro activity is required to maintain glomerular macromolecular permeability, albumin permeability (P(alb)) was studied after incubation of glomeruli from normal animals with a series of monoclonal (mAb) and polyclonal antibodies. Reagents included mAbs to rabbit and rat PTPro and polyclonal rabbit immune IgG to rat PTPro. mAb 4C3, specific to the amino acid core of PTPro, decreased its phosphatase activity and increased P(alb) of rabbit glomeruli in a time- and concentration-dependent manner. In contrast, mAb P8E7 did not diminish phosphatase activity and did not alter P(alb). Preincubation of 4C3 with PTPro extracellular domain fusion protein blocked glomerular binding and abolished permeability activity. In parallel experiments, P(alb) of rat glomeruli was increased by two mAbs (1B4 and 1D1) or by polyclonal anti-rat PTPro. We conclude that PTPro interaction with specific antibodies acutely increases P(alb). The identity of the normal ligand for PTPro and of its substrate, as well as the mechanism by which phosphatase activity of this receptor affects the filtration barrier, remain to be determined.
Asunto(s)
Albúminas/metabolismo , Anticuerpos Monoclonales/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Glomérulos Renales/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/inmunología , Albuminuria/metabolismo , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Tasa de Filtración Glomerular/efectos de los fármacos , Glomérulos Renales/efectos de los fármacos , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína/efectos de los fármacos , Conejos , Ratas , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Factores de TiempoRESUMEN
Because loss of podocytes associates with glomerulosclerosis, monitoring podocyte loss by measuring podocyte products in urine may be clinically useful. To determine whether a single episode of podocyte injury would cause persistent podocyte loss, we induced limited podocyte depletion using a diphtheria toxin receptor (hDTR) transgenic rat. We monitored podocyte loss by detecting nephrin and podocin mRNA in urine particulates with quantitative reverse transcriptase-PCR. Aquaporin 2 mRNA served as a kidney reference gene to account for variable kidney contribution to RNA amount and quality. We found that a single injection of diphtheria toxin resulted in an initial peak of proteinuria and podocyte mRNAs (podocin and nephrin) followed 8 d later by a second peak of proteinuria and podocyte mRNAs that were podocin positive but nephrin negative. Proteinuria that persisted for months correlated with podocin-positive, nephrin-negative mRNAs in urine. Animals with persistent podocyte mRNA in urine progressed to ESRD with global podocyte depletion and interstitial scarring. Podocytes in ectatic tubules expressed podocalyxin and podocin proteins but not nephrin, compatible with detached podocytes' having an altered phenotype. Parallel human studies showed that biopsy-proven glomerular injury associated with increased urinary podocin:aquaporin 2 and nephrin:aquaporin 2 molar ratios. We conclude that a single episode of podocyte injury can trigger glomerular destabilization, resulting in persistent podocyte loss and an altered phenotype of podocytes recovered from urine. Podocyte mRNAs in urine may be a useful clinical tool for the diagnosis and monitoring of glomerular diseases.
Asunto(s)
Necrosis de la Corteza Renal/patología , Enfermedades Renales/genética , Enfermedades Renales/patología , Glomérulos Renales/patología , Podocitos/patología , ARN Mensajero/orina , Animales , Animales Modificados Genéticamente , Acuaporina 2/genética , Biomarcadores , Progresión de la Enfermedad , Femenino , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Necrosis de la Corteza Renal/genética , Fallo Renal Crónico/genética , Fallo Renal Crónico/patología , Masculino , Proteinuria/genética , Proteinuria/patología , ARN Mensajero/genética , Ratas , Valores de ReferenciaRESUMEN
The right kidney of anesthetized rats was imaged with intermittent diagnostic ultrasound (1.5 MHz; 1-s trigger interval) under exposure conditions simulating those encountered in human perfusion imaging. The rats were infused intravenously with 10 microL/kg/min Definity (Bristol-Myers Squibb Medical Imaging, Inc., N. Billerica, MA, USA) while being exposed to mechanical index (MI) values of up to 1.5 for 1 min. Suprathreshold MI values ruptured glomerular capillaries, resulting in blood filling Bowman's space and proximal convoluted tubules of many nephrons. The re-establishment of a pressure gradient after hemostasis caused the uninjured portions of the glomerular capillaries to resume the production of urinary filtrate, which washed some or all of the erythrocytes out of Bowman's space and cleared blood cells from some nephrons into urine within six hours. However, many of the injured nephrons remained plugged with tightly packed red cell casts 24 h after imaging and also showed degeneration of tubular epithelium, indicative of acute tubular necrosis. The additional damage caused by the extravasated blood amplified that caused by the original cavitating gas body. Human nephrons are virtually identical to those of the rat and so it is probable that similar glomerular capillary rupture followed by transient blockage and/or epithelial degeneration will occur after clinical exposures using similar high MI intermittent imaging with gas body contrast agents. The detection of blood in postimaging urine samples using standard hematuria tests would confirm whether or not clinical protocols need to be developed to avoid this potential for iatrogenic injury.
Asunto(s)
Medios de Contraste/toxicidad , Hemorragia/etiología , Enfermedades Renales/etiología , Riñón/diagnóstico por imagen , Microburbujas/efectos adversos , Nefronas/lesiones , Animales , Hematuria/etiología , Hemorragia/patología , Riñón/ultraestructura , Enfermedades Renales/patología , Ratas , Ultrasonografía/efectos adversosRESUMEN
One kidney of anesthetized rats was imaged by diagnostic ultrasound with contrast agent under conditions simulating both the geometry and the attenuation encountered during human perfusion imaging. Contrary to earlier predictions, glomerular capillary rupture with blood loss into Bowman's space and proximal tubules occurred in our clinically relevant model system. Quantitative analysis of histologic sections showed that 37 +/- 5% of the glomeruli at the center of the scan plane had blood cells in Bowman's space after imaging for 1 min with 1.8 MPa (mechanical index equivalent, MIe = 1.5) with a 1 s image trigger interval during IV injection of 10 microl/kg/min of Definity contrast agent (as recommended by the manufacturer). This percentage decreased rapidly with decreasing peak rarefactional pressure amplitude to an apparent threshold of 0.73 MPa (MIe = 0.6). The percentage of glomeruli with hemorrhage decreased in proportion to dose when reduced below the recommended value, but leveled-off at doses above it. The percentage of glomerular hemorrhage increased with increasing numbers of image exposures, with an initial rate of 1.1% per image. The glomerular hemorrhage also depended on the frame trigger interval with no hemorrhage evident for continuous imaging but a maximal effect for trigger intervals greater than about 1 s. These results indicated that there is a potential for clinical diagnostic ultrasound with contrast agent to induce glomerular hemorrhage.
Asunto(s)
Medios de Contraste/efectos adversos , Aumento de la Imagen , Riñón/diagnóstico por imagen , Animales , Capilares , Medios de Contraste/administración & dosificación , Relación Dosis-Respuesta a Droga , Hemorragia/etiología , Riñón/anatomía & histología , Glomérulos Renales/anatomía & histología , Glomérulos Renales/efectos de los fármacos , Microburbujas , Modelos Animales , Fantasmas de Imagen , Ratas , UltrasonografíaRESUMEN
Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCepsilon1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif-containing GTPase-activating protein 1 as a new interaction partner of PLCepsilon1. Two siblings with a missense mutation in an exon encoding the PLCepsilon1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.
Asunto(s)
Mutación , Síndrome Nefrótico/enzimología , Síndrome Nefrótico/genética , Fosfolipasas de Tipo C/genética , Animales , Niño , Preescolar , Clonación Molecular , Modelos Animales de Enfermedad , Femenino , Marcación de Gen , Genes Recesivos , Homocigoto , Humanos , Lactante , Riñón/enzimología , Riñón/patología , Masculino , Modelos Genéticos , Mutación Missense , Síndrome Nefrótico/tratamiento farmacológico , Síndrome Nefrótico/patología , Fosfoinositido Fosfolipasa C , Ratas , Eliminación de Secuencia , Pez Cebra/genéticaRESUMEN
Biologic aging is accelerated by high-calorie intake, increased free radical production, and oxidation of key biomolecules. Fischer 344 rats that are maintained on an ad libitum diet develop oxidant injury and age-associated glomerulosclerosis by 24 mo. Calorie restriction prevents both oxidant injury and glomerulosclerosis. Ceruloplasmin (Cp) is a copper-containing ferroxidase that functions as an antioxidant in part by oxidizing toxic ferrous iron to nontoxic ferric iron. Glomerular Cp mRNA and protein expression were measured in ad libitum-fed and calorie-restricted rats at ages 2, 6, 17, and 24 mo. In ad libitum-fed rats, Cp mRNA expression increased six-fold (P < 0.01) and protein expression increased five-fold (P = 0.01) between 2 and 24 mo of age. In calorie-restricted rats, Cp mRNA expression increased three-fold (P < 0.01) and protein expression increased 1.6-fold (NS) between 2 and 24 mo of age. Both the cell-associated alternately spliced variant and secreted variants of Cp were expressed. Immunofluorescent analysis showed that Cp was expressed by the parietal epithelial cells that line the inner aspect of Bowman's capsule in the glomerulus. Cp also was present in urine, particularly of old ad libitum-fed rats with high tissue Cp expression. Cp expression by Bowman's capsule epithelial cells therefore occurred in direct proportion to known levels of oxidant activity (older age and high-calorie diet) and is secreted into the urine. It is suggested that Cp expression at this site may be part of the repertoire of the glomerular parietal epithelial cell to protect the glomerular podocytes and the downstream nephron from toxic effects of filtered molecules, including ferrous iron.
Asunto(s)
Envejecimiento/metabolismo , Ceruloplasmina/metabolismo , Ingestión de Energía/fisiología , Células Epiteliales/metabolismo , Glomérulos Renales/metabolismo , Animales , Antioxidantes/metabolismo , Células Cultivadas , Ceruloplasmina/orina , Ratas , Ratas Endogámicas F344RESUMEN
Whether podocyte depletion could cause the glomerulosclerosis of aging in Fischer 344 rats at ages 2, 6, 17, and 24 mo was evaluated. Ad libitum-fed rats developed proteinuria and glomerulosclerosis by 24 mo, whereas calorie-restricted rats did not. No evidence of age-associated progressive linear loss of podocytes from glomeruli was found. Rather, ad libitum-fed rats developed glomerular enlargement over time. To accommodate the increased glomerular volume, podocytes principally underwent hypertrophy, whereas other glomerular cells underwent hyperplasia. Stages of hypertrophy through which podocytes pass en route to podocyte loss and glomerulosclerosis were identified: Stage 1, normal podocyte; stage 2, nonstressed podocyte hypertrophy; stage 3, "adaptive" podocyte hypertrophy manifest by changes in synthesis of structural components (e.g., desmin) but maintenance of normal function; stage 4, "decompensated" podocyte hypertrophy relative to total glomerular volume manifest by reduced production of key machinery necessary for normal podocyte function (e.g., Wilms' tumor 1 protein [WT1], transcription factor pod1, nephrin, glomerular epithelial protein 1, podocalyxin, vascular endothelial growth factor, and alpha5 type IV collagen) and associated with widened foot processes and decreased filter efficiency (proteinuria); and stage 5, podocyte numbers decrease in association with focal segmental glomerulosclerosis. In contrast, in calorie-restricted rats, glomerular enlargement was minor, significant podocyte hypertrophy did not occur, podocyte machinery was unchanged, there was no proteinuria, and glomerulosclerosis did not develop. Glomerular enlargement therefore was associated with podocyte hypertrophy rather than hyperplasia. Hypertrophy above a certain threshold was associated with podocyte stress and then failure, culminating in reduced podocyte numbers in sclerotic glomeruli. This process could be prevented by calorie restriction.
Asunto(s)
Restricción Calórica , Glomeruloesclerosis Focal y Segmentaria/etiología , Glomeruloesclerosis Focal y Segmentaria/prevención & control , Glomérulos Renales/patología , Podocitos/patología , Adaptación Fisiológica , Factores de Edad , Animales , Recuento de Células , Ingestión de Energía , Hipertrofia , Podocitos/fisiología , Ratas , Ratas Endogámicas F344RESUMEN
Glomerular injury and proteinuria in diabetes (types 1 and 2) and IgA nephropathy is related to the degree of podocyte depletion in humans. For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed. The rodent homologue does not act as a diphtheria toxin (DT) receptor, thereby making rodents resistant to DT. Injection of DT into transgenic rats but not wild-type rats resulted in dose-dependent podocyte depletion from glomeruli. Three stages of glomerular injury caused by podocyte depletion were identified: Stage 1, 0 to 20% depletion showed mesangial expansion, transient proteinuria and normal renal function; stage 2, 21 to 40% depletion showed mesangial expansion, capsular adhesions (synechiae), focal segmental glomerulosclerosis, mild persistent proteinuria, and normal renal function; and stage 3, >40% podocyte depletion showed segmental to global glomerulosclerosis with sustained high-grade proteinuria and reduced renal function. These pathophysiologic consequences of podocyte depletion parallel similar degrees of podocyte depletion, glomerulosclerosis, and proteinuria seen in diabetic glomerulosclerosis. This model system provides strong support for the concept that podocyte depletion could be a major mechanism driving glomerulosclerosis and progressive loss of renal function in human glomerular diseases.